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Federal Democratic 
Republic of Ethiopia 


In order to promote public education and public safety, 
equal justice for all, a better informed citizenry, the rule 
of law, world trade and world peace, this legal document is 
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the right of all humans to know and speak the laws that 
govern them. 

ET ISO 5508 (1990) (English) : Animal 
and vegetable fats and oils — Analysis 
by gas chromatography of methyl esters 
of fatty acids 

ISO inside' 

* •♦ ♦ *«*A#^** *T* 






ETHIOPIAN ES ISO 5508 :201 2 


First edition 

Animal and vegetable fats and oils - 
Analysis by gas chromatography of 
methyl esters of fatty acids 

(Identical with ISO 5508:1990) 

ICS: 67.200.10 

Published by Ethiopian Standards Agency 


This Ethiopian Standard has been prepared under the direction of the Technical Committee for Animal 
and vegetables fats and oils (TC 23) and published by the Ethiopian Standards Agency (ESA). 

The standard is identical with ISO :19 , 

published by International Organization for Standardization (ISO). 

For the purpose of this Ethiopian Standard, the adopted text shall be modified as follows. 

• The phrase "International Standard" shall be read as "Ethiopian Standard"; and 

• A full stop (.) shall substitute comma (,) as decimal marker. 


ES ISO 5508:2012 

Animal and vegetable fats and oils — Analysis by gas 
chromatography of methyl esters of fatty acids 

1 Scope 

This International Standard gives general guidance 
for the application of gas chromatography, using 
packed or capillary columns, to determine the qual- 
itative and quantitative composition of a mixture of 
fatty acid methyl esters obtained in accordance with 
the method specified in ISO 5509. 

The method is not applicable to polymerized fatty 

3.2.2 Air or oxygen, free from organic impurities. 

3.3 Reference standard 

A mixture of methyl esters of pure fatty acids, or the 
methyl esters of a fat of known composition, prefer- 
ably similartothat of the fatty matter to be analysed. 

Care shall be taken to prevent the oxidation of 
polyunsaturated fatty acids. 

2 Normative reference 

The following standard contains provisions which, 
through reference in this text, constitute provisions 
of this International Standard. At the time of publi- 
cation, the edition indicated was valid. All standards 
are subject to revision, and parties to agreements 
based on this International Standard are encour- 
aged to investigate the possibility of applying the 
most recent edition of the standard indicated below. 
Members of lEC and ISO maintain registers of cur- 
rently valid International Standards. 

ISO 5509:1978, Animal and vegetable fats and oils — 
Preparation of methyl esters of fatty acids. 

3 Reagents 

3.1 Carrier gas 

Inert gas (nitrogen, helium, argon, hydrogen, etc.), 
thorougly dried and with an oxygen content of less 
than 10 mg/kg. 

NOTE 1 Hydrogen, which is used as a carrier gas only 
with capillary columns, can double the speed of analysis 
but is hazardous. Safety devices are available. 

3.2 Auxiliary gases 

3.2.1 Hydrogen (purity ^ 99,9 %), free from or- 
ganic impurities. 

4 Apparatus 

The Instructions given relate to the usual equipment 
used for gas chromatography, employing packed 
and/or capillary columns and a flame-ionization de- 
tector. Any apparatus giving the efficiency and re- 
solution specified in 5.1.2 is suitable. 

4.1 Gas chromatograph. 

The gas chromatograph shall comprise the following 

4.1.1 Injection system. 

Use an injection system either 

a) with packed columns, having the least dead- 
space possible (in this case the injection system 
shall be capable of being heated to a temper- 
ature 20 °C to 50 °C higher than that of the col- 
umn), or 

b) with capillary columns, in which case the in- 
jection system shall be specially designed for 
use with such columns. It may be of the split type 
or it may be of the splitless on column injector 

NOTE 2 In the absence of fatty acids with less than 
16 carbon atoms, a moving needle injector may be 


ES ISO 5508:2012 

4.1.2 Oven. 

The oven shall be capable of heating the column to 
a temperature of at least 260 °C and of maintaining 
the desired temperature to within 1 °C with a packed 
column and within 0,1 °C with a capillary column. 
The last requirement is particularly important when 
a fused silica tube is used. 

The use of temperature-programmed heating is re- 
commended in all cases, and In particular for fatty 
acids with less than 16 carbon atoms. 

4.1.3 Packed column. Column, constructed of a material inert to 
the substances to be analysed (i.e. glass or stain- 
less steel) having the following dimensions. 

a) Length: 1 m to 3 m. A relatively short column 
should be used when long-chain fatty acids 
(above C20) are present. When analysing acids 
with 4 or 6 carbon atoms, it is recommended that 
a column 2 m in length is used. 

b) Internal diameter: 2 mm to 4 mm. 

3 If polyunsaturated components with more than three 
double bonds are present, they may be decomposed In a 
stainless steel column. 

4 A system with packed twin columns may be used. Packing, comprising the following elements. 

a) Support: Acid-washed and silanized diatom- 
aceous earth, or other suitable inert support with 
a narrow range of grain size (25 \xrr\ range be- 
tween the limits 125 p,m to 200 ^m), the average 
grain size being related to the internal diameter 
and length of the column. 

b) Stationary phase: Polyester type of polar liquid 
(e.g. diethylene glycol polysuccinate, butanediol 
polysuccinate, ethyleneglycol polyadipate, etc.), 
cyanosilicones or any other liquid permitting the 
chromatographic separation required (see 
clause 5). The stationary phase should amount 
to 5 % {m/m) to 20 % (m/m) of the packing. A 
non-polar stationary phase can be used for cer- 
tain separations. Conditioning of the column. 

With the column disconnected, if possible, from the 
detector, gradually heat the oven to 185 *^C and pass 
a current of Inert gas through the freshly prepared 
column at a rate of 20 ml/min to 60 ml/min for at 
least 16 h at this temperature, and for a further 2 h 
at 195 X. 

4.1.4 Capillary column. Tube, made of a material inert to the sub- 
stances to be analysed (usually glass or fused 
silica). The Internal diameter shall be between 
0,2 mm and 0,8 mm. The internal surface shall un- 
dergo an appropriate treatment (e.g. surface prepa- 
ration, inactivation) before receiving the stationary 
phase coating. A length of 25 m is sufficient in most 
cases. Stationary phase, usually of the type 
polyglycol [poly(ethylene glycol) 20000], polyester 
(butanediol polysuccinate) or polar polysiloxane 
(cyanosilicones). Bonded (cross-linked) columns are 

NOTE 5 There is a risk of polar polyslloxanes giving rise 
to difficulties in the identification and separation of 
linolenic acid and C20 acids. 

The coatings shall be thin, i.e. 0,1 \xm to 0,2 jim. Assembly and conditioning of the column. 

Observe the normal precautions for assembling 
capillary columns, i.e. arrangement of the column in 
the oven (support), choice and assembly of joints 
(leak tightness), positioning of the ends of the col- 
umn in the injector and the detector (reduction of 
dead-spaces). Place the column under a flow of 
carrier gas [e.g. 0,3 bar (30 kPa) for a column of 
length 25 mm and internal diameter 0,3 mm]. 

Condition the column by temperature programming 
of the oven at 3 ''C/min from ambient temperature 
to a temperature 10 °C below the decomposure limit 
of the stationary phase. Maintain the oven at this 
temperature for 1 h until stabilization of the base- 
line. Return it to 180 °C to work under isothermal 

NOTE 6 Suitably pre-conditioned columns are available 

4.1.5 Detector, preferably capable of being heated 
to a temperature above that of the column. 

4.2 Syringe. 

The syringe shall have a maximum capacity of 
10 |il, and be graduated in 0,1 ^il divisions. 

4.3 Recorder. 

If the recorder curve is to be used to calculate the 
composition of the mixture analysed, an electronic 
recorder of high precision, compatible with the ap- 
paratus used, is required. The recorder shall have 
the following characteristics: 

a) rate of response, below 1,5 s, preferably 1 s (the 
rate of response is the time taken for the re- 


ES ISO 5508:2012 

cording pen to pass from % to 90 % following 
the sudden introduction of a 100 % signal); 

b) width of the paper, 20 cm minimum; 

c) paper speed, adjustable to values between 
0,4 cm/min and 2,5 cm/min. 

4.4 integrator or calculator (optional). 

Rapid and accurate calculation can be performed 
with the help of an electronic integrator or calcula- 
tor. This shall give a linear response with adequate 
sensitivity, and the correction for deviation of the 
base-line shall be satisfactory. 

5 Procedure 

The operations described In 5.1 to 5.3 relate to the 
use of a flame-ionization detector. 

As an alternative a gas chromatograph employing 
a catharometer detector (working on the principle 
of thermal conductivity changes) may be used. The 
operating conditions are then modified as described 
in clause 7. 

5,1 Test conditions 

5.1.1 Selection of optimum operating conditions Packed column 

In the selection of the test conditions, the following 
variables should be taken into account: 

a) the length and diameter of the column; 

b) the nature and amount of the stationary phase; 

c) the temperature of the column; 

d) the carrier gas flow; 

e) the resolution required; 

f) the size of the test portion, selected in such a 
way that the assembly of the detector and 
electrometer gives a linear response; 

g) the duration of analysis. 

In general, the values given in table 1 and table 2 will 
lead to the desired results, i.e. at least 2 000 theore- 
tical plates per metre of column length for methyl 
stearate and its elution within about 15 min. 

Where the apparatus allows it, the injector should 
be at a temperature of about 200 ^C and the detector 

at a temperature equal to or higher than that of the 

As a rule, the ratio of the flow-rate of the hydrogen 
supplied to the flame-ionization detector to that of 
the carrier gas varies from 1:2 to 1:1 depending on 
the diameter of the column. The flow of oxygen is 
about 5 to 10 times that of the hydrogen. 

Table 1 

Internal diameter 
of column 


Carrier gas flow 



15 to 25 
20 to 40 
40 to 60 

Table 2 

of stationary phase 

% (m/m) 

Column temperature 




5.1 .1 .2 Capillary column 

The properties of efficiency and permeability of 
capillary columns mean that the separation between 
constituents and the duration of the analysis are 
largely dependent on the flow-rate of the carrier gas 
in the column. It will therefore be necessary to opti- 
mize the operating conditions by acting on this pa- 
rameter (or more simply on the headloss of the 
column), according to whether one wishes to im- 
prove the separations or to make a rapid analysis. 

5.1.2 Determination of the number of theoretical 
plates (efficiency) and resolution 

(See figure!) 

Carry out the analysis of a mixture of methyl 
stearate and methyl oleate In about equivalent pro- 
portions (for example, methyl esters from cocoa 

Choose the temperature of the column and the car- 
rier gas flow so that the maximum of the methyl 
stearate peak is recorded about 15 min after the 
solvent peak. Use a sufficient quantity of the mixture 
of methyl esters that the methyl stearate peak oc- 
cupies about three-quarters of the full scale. 


ES ISO 5508:2012 

Calculate the number of theoretical plates, n (effi- 
ciency), using the formula 





The operating conditions to be selected are those 
which will afford at least 2000 theoretical plates per 
metre of column length for methyl stearate and a 
resolution of at least 1,25. 

and the resolution, R, using the formula 


C0(,) + a>(„) 


rfr(i) is the retention distance, in millimetres, 
from the start of the chromatogram to the 
maximum of the peak for methyl 

co(,j and a>(,,) are the widths, in millimetres, of 
the peaks for methyl stearate and methyl 
oleate respectively, measured between 
the points of intersection of the tangents 
at the points of inflexion of the curve with 
the base-line; 

A is the distance, in millimetres, between 
the peak maxima for methyl stearate and 
methyl oleate. 

5.2 Test portion 

Using the syringe (4.2), take 0,1 \i\ to 2 |il of the sol- 
ution of methyl esters prepared according to 
ISO 5509 and inject them into the column. 

In the case of esters not in solution, prepare a sol- 
ution of approximately 100 mg/ml in heptane of 
chromatographic quality, and inject 0,1 \x\ to 1 [il of 
this solution. 

If the analysis Is for constitutents present only in 
trace amounts, the size of the test portion may be 
increased (up to ten-fold). 

5.3 Analysis 

Generally, the operating conditions shall be those 
defined in 5.1.1 








Figure 1 — Chromatogram for determining tlie number of theoretical plates (efficiency) and resolution 


ES ISO 5508:2012 

Nevertheless, it is possible to work with a lower 
column temperature when the determination of fatty 
acids with fewer than 12 carbon atoms is required, 
or at a higher temperature when determining fatty 
acids with more than 20 carbon atoms. On occasion, 
it is possible to employ temperature programming 
in both these cases. For example, if the sample 
contains the methyl esters of fatty acids with fewer 
than 12 carbon atoms. Inject the sample at 100 ''C 
(or at 50 °C to 60 ^C If butyric acid is present) and 
immediately raise the temperature at a rate of 
4 °C/min to 8 ""C/min to the optimum. In certain 
cases, the two procedures can be combined. 

After the programmed heating, continue the elution 
at a constant temperature until all the components 
have been eluted. If the instrument does not have 
programmed heating, use it at two fixed temper- 
atures between 100 °C and 195 ^C. 

If necessary, It is recommended that an analysis be 
carried out on two fixed phases with different polar- 
ities to verify the absence of masked peaks, for ex- 
ample for fish oils or In the case of the simultaneous 
presence of C^s-^ and CgoiO' o^ ^18:3 si^d Ci8:2 con- 

5.4 Preparation of the reference 
chromatogram and reference graphs 

Analyse the reference standard mixture (3.3), using 
the same operating conditions as those employed 
for the sample, and measure the retention times or 
retention distances for the constituent fatty acids. 
Construct on semi-logarithmic paper, for any degree 
of unsaturation, the graphs showing the logarithm 
of retention time or distance as a function of the 
number of carbon atoms. In isothermal conditions, 
the graphs for straight-chain acids of the same de- 
gree of unsaturation should be straight lines. These 
lines should be approximately parallel. 

It Is necessary to avoid conditions such that 
"masked peaks" exist, i.e. where the resolution Is 
insufficient to separate two constituents. 

6 Expression of results 

6.1 Qualitative analysis 

Identify the methyl ester peaks for the sample from 
the graphs prepared in 5.4, if necessary by interpo- 

6.2 Quantitative analysis 

6.2.1 Determination of the composition 

Apart from exceptional cases, use the internal nor- 
malization method, i.e. assume that the whole of the 
components of the sample are represented on the 

chromatogram, so that the total of the areas under 
the peaks represents 100 % of the constituents (total 

If the equipment includes an integrator, use the fig- 
ures obtained therefrom. If not, determine the area 
under each peak by multiplying the height of the 
peak by its width at mid-height, and where neces- 
sary take Into account the various attenuations used 
during the recording. 

6.2.2 l\Aethod of calculation General case 

Calculate the content of a given component /, ex- 
pressed as a percentage by mass of methyl esters, 
by determining the percentage represented by the 
area of the corresponding peak relative to the sum 
of the areas of all the peaks, using the following 





A^ is the area under the peak corresponding 
to component /; 

Y.A Is the sum of the areas under all the 

Give the result to one decimal place. 

NOTE 7 In this general case, the result of the calcu- 
lation based on relative areas is considered to represent 
a percentage by mass. For the cases in which this as- 
sumption is not allowed, see Use of correction factors 

In certain cases, for example in the presence of fatty 
acids with fewer than 8 carbon atoms or of acids 
with secondary groups, when using thermal con- 
ductivity detectors or where the highest degree of 
accuracy Is particularly required, correction factors 
should be used to convert the percentages of peak 
areas Into mass percentages of the components. 

Determine the correction factors with the help of a 
chromatogram derived from the analysis of a refer- 
ence mixture of methyl esters of known composition, 
carried out under operating conditions identical with 
those used for the sample. 

For this reference mixture, the percentage by mass 
of component / is given by the formula 





ES ISO 5508:2012 


m^ Is the mass of component / in the refer- 
ence mixture; 

Y/n Is the total of the masses of the various 
components of the reference mixture. 

From the chromatogram of the reference mixture 
(5.4), calculate the percentage (area/area) for com- 
ponent / as follows: 




A^ is the area under the peak corresponding 
to component i\ 

YA is the sum of the areas under all the 

The correction factor is then calculated as 


m^ X Va 

A, X Y^m 

Commonly, the correction factors are expressed 
relative to /<^ci6» so that the relative factors become 



For the sample, the content of each component /, 
expressed as a percentage by mass of methyl 
esters, is 

K\ X A. 

' X 100 

Give the results to one decimal place. Use of an internal standard 

In certain analyses (for example where not all of the 
fatty acids are quantified, such as when acids with 
4 and 6 carbons are present alongside adds with 16 
and 18 carbons, or when it is necessary to deter- 
mine the absolute amount of a fatty acid in a sam- 
ple) it is necessary to use an internal standard. Fatty 
acids with 5, 15 or 17 carbons are frequently used. 
The correction factor (if any) for the internal stan- 
dard should be determined. 

The percentage by mass of component /, expressed 
as methyl esters, is then given by the formula 

m^ X K\ X Aj 
mxK\x A^ 



A^ is the area under the peak corresponding 
to component /; 

A^ is the area under the peak corresponding 
to the internal standard; 

K\ is the correction factor for component / 
(relative to ATcie); 

K\ is the correction factor for the internal 
standard (relative to Kq^^Y 

m is the mass, in milligrams, of the test 

/TZg is the mass, in milligrams, of the internal 

Give the results to one decimal place. 

6.2.3 Precision 

The values for repeatability and reproducibility given 
below cover the preparation of the methyl esters 
according to ISO 5509, together with the gas chro- 
matographic analysis described in this International 
Standard. The figures have been accepted histor- 
ically. Repeatability 

The difference between the values of two determi- 
nations, carried out in rapid succession by the same 
operator using the same apparatus on the same test 
sample and for constituents present in excess of 
5 % (mjm), shall not exceed 3 % (relative) of the 
determined value, with a maximum of 1 % (mjm). 
For constituents present in smaller quantities, the 
difference shall not exceed a value of 0,2 % (m/m). Reproducibility 

The difference between the values of the final result 
obtained by two different laboratories, using this 
method for the analysis of the same laboratory 
sample for constituents present in excess of 
5 % {m/m), shall not exceed 10 % (relative) of the 
determined value, with a maximum of 3 % (mjm). 
For constituents present in smaller quantities, this 
difference shall not exceed a value of 0,5 % (mjm). 

7 Special case — Use of a catharometer 
detector (working on the principle of 
thermal conductivity changes) 

A gas chromatograph employing a detector working 
on the principle of thermal conductivity changes (a 
catharometer) may also be used for the determi- 
nation of the qualitative and quantitative composi- 
tion of a mixture of fatty acid methyl esters. If it is 


ES ISO 5508:2012 

used, the conditions specified in clause 4 and 
clause 5 should be modified as shown in table 3. 

For quantitative analysis, use the correction factors 
defined in 

Table 3 




Length: 2 m to 4 m 

Internal diameter: 4 mm 


Grain size between 160 ^m 
and 200 nm 

Concentration of sta- 
tionary phase 

15 % (m/m) to 25 % (m/m) 

Carrier gas 

Helium or, failing this, hydro- 
gen, with as low an oxygen 
content as possible 

Auxiliary gases 


Injector temperature 

From 40 '^'C to 60 °C above that 
of the column 

Column temperature 


Flow of carrier gas 

Usually between 60 ml/min 
and 80 ml/min 

Size of test portion 

Usually between 0,5 ^1 and 

8 Test report 

The test report shall specify the methods used for 
the preparation of the methyl esters and for the gas 
chromatographic analysis, and the results obtained. 
It shall also mention all operating details not speci- 
fied in this International Standard, or regarded as 
optional, together with details of any incidents which 
may have influenced the results. 

The test report shall include all information neces- 
sary for the complete identification of the sample. 

























Organization and Objectives 

sroTOue to 1 

The Ethiopian Standards Agency (ESA) is the national standards bod^^MRiiopia 
estabHshed in 2010 based on regulation No. 193/2010.ESA is establisroTOue to the 
restructuring of Quality and Standards Authority of Ethiopia (QiAELwhich was 
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Ethiopian Standard 


^rj ^tionai m arKet. 
)nal technSRbmr 

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